Not Applicable 
Not Applicable
1. Field of the Invention
The present invention pertains generally to three-dimensional optical microscopy, and more particularly to a method and apparatus for three-dimensional optical microscopy which employs dual opposing objective lenses about a sample to obtain a high level of depth resolution.
2. Description of the Background Art
Optical microscopy has experienced a remarkable renaissance in the medical and biological sciences during the last decade. The increased importance of optical microscopy has been due to new developments in fluorescent probe technology, and the availability of quantitative three-dimensional image data obtained through either computational deconvolution or scanning confocal microscopy.
Optical microscopy offers several advantages over non-optical microscopy techniques. Use of optical microscopy allows viewing of living tissue samples in their natural state. Electron microscopy, in comparison, requires microscopy samples which are dried and exposed to vacuum. Additionally, the interior of the sample can be viewed and mapped in three dimensions using optical microscopy, whereas scanning electron microscopy and other scanned probe microscopies map only the surface of the is sample, and thus cannot provide information about the sample interior. Yet another advantage of optical microscopy is that particular cellular components can be recognized and mapped out with great specificity by staining with fluorescent probes. It is now possible to synthesize fluorescent probes with specificity for nearly any given biomolecule.
The only important drawback to optical microscopy is its limited resolution, which is related to the angle over which the objective lens can collect light, and ultimately from the finite wavelength of light. Thus, any technology such as the present invention that significantly increases the resolution of optical microscopy will have important applications in cellular biology, medical imaging, and other biotechnology fields.
Presently there are two primary approaches to three-dimensional optical microscopy: optical sectioning microscopy, which is also known as computational deconvolution, and scanning confocal microscopy.
In optical sectioning microscopy, a series of images of the microscopy sample are acquired, with the focus moved successively through sections of the sample to obtain successive images. Each image contains in-focus information from the parts or sections of the sample which are in the focal plane, and blurred, out-of-focus information from the other parts of the sample. Analysis of the entire data set by computer allows reconstruction of the three-dimensional structure of the sample. The reconstruction process employs computational algorithms and a previously stored reference data set describing the blur caused by a single point source. Optical sectioning microscopy is a xe2x80x9cwidefieldxe2x80x9d microscopy in which large area images are recorded, typically by a charge-coupled device array (CCD) camera. Thus, high light throughput and high data acquisition speeds are possible with this technique.
In confocal microscopy, a focused laser beam is used as a light source, and light is detected by a photomultiplier tube through a pinhole which is focused onto the same spot in the sample as the laser. This combined focal point is then scanned in three dimensions through the sample, and the detected intensity as a function of spot position is used to obtain a three-dimensional image of the sample. The pinhole partially suppresses out-of-focus information and improves the resolution, but at the cost of discarding much of the light. This loss of light necessitates long exposure times, which makes operation slow and often causes severe sample bleaching problems. Confocal microscopy operations are further slowed down by the fact that the data pixels are acquired one at a time, as opposed to up to a million in parallel for the large area imaging employed in optical sectioning microscopy.
Both optical sectioning microscopy and confocal microscopy suffer an important drawback in that the depth resolution or Z-direction resolution is several times worse than that in the transverse, or XY, plane. The limitation on Z-direction resolution is caused by fundamental geometrical limitations which are discussed in detail below. The present invention provides a method and apparatus for optical microscopy in which the Z-resolution is not only equal to that of the resolution in the XY plane, but is increased to more than double the resolution in the XY plane obtained heretofore with optical sectioning microscopy. This increase in Z-direction resolution is achieved by the present invention while also maintaining the high light throughput and data acquisition speeds available through optical sectioning microscopy.
There are two previously known optical microscopy methods which employ dual opposing objective lenses. One method, which is known as 4Pi Confocal Microscopy, is a confocal, rather than a widefield, microscopic method. 4Pi Confocal Microscopy can generally be employed in three ways. In a first mode, focused laser light is used to illuminate a sample from both objective lenses and interfere in the sample. In a second mode the emitted light is collected from both directions and combined onto a single pinhole detector. The third mode involves the combination of the first two modes simultaneously. Being a confocal technique, however, all modes of 4Pi Confocal Microscopy have poor light throughput and lengthy data acquisition times due to loss of light caused by the pinhole photodector  photodetector and the slowness of the pixel-by-pixel data acquisition.
The second known optical microscopy method which employs two opposing lenses is generally called Standing Wave Fluorescence Microscopy (SWFM). This technique requires a light source with great temporal and spatial coherence, typically in the form of a laser. The spatially and temporally coherent light source results in an interference pattern in sample space which is a sinusoidal standing wave (hence the name) that extends throughout the observed region of the sample.
SWFM could in principle achieve similar Z resolution as one embodiment of the present invention (the I3M embodiment described herein) but only by combining several different standing wave patterns in sequence through use of scanning mirrors on similar dynamic devices, or by using multiple individually coherent but mutually incoherent light sources, such as a plurality of lasers. The present invention provides the increased Z-direction resolution without requiring such moving parts, requires only a single, spatially incoherent light source such as an arc lamp or incandescent bulb, and does not require temporal coherence beyond that exhibited by any band-limited light source. The use of a simple incoherent light source allows free choice of wavelength of the illumination light, while lasers are available in only a limited selection of wavelengths. Furthermore, one embodiment of the present invention (the I5M embodiment described herein) achieves greater Z resolution than is possible through SWFM alone.
Thus, the present invention differs from, and has advantages compared to, all previously known 3D microscopy techniques. Compared to any mode of microscopy that uses a single objective lens, the present invention offers higher Z resolution. Compared to SWFM, the present invention uses simpler illumination means and offers a greater selection of illumination wavelengths, and in one of its embodiments offers higher Z resolution. Compared to 4Pi Confocal Microscopy, the present invention offers simpler illumination means, a greater selection of illumination wavelengths, greater data acquisition speed, and more efficient use of observed or emitted light, which can lead to less severe sample bleaching.
Thus, there is a need for a method and apparatus for three-dimensional optical microscopy which provides greatly enhanced depth or Z-direction resolution, which has a high light throughput, which has a high data acquisition speed, and which does not require use of spatially coherent light sources. The present invention satisfies these needs, as well as others, and generally overcomes the deficiencies found in known optical microscopy devices and methods.
The present invention generally pertains to a method and apparatus for three-dimensional optical microscopy which employs dual opposing objective lenses about a sample. There are three preferred embodiments of the invention which, employing essentially the same apparatus, allow the sample to be illuminated from one or both objective lenses, and to be observed and recorded through one or both objective lenses.
By way of example and not of limitation, the present invention generally includes first and second objective lenses which are mounted opposite to each other about a thin sample, with at least one of the objective lenses including translational adjustment means. Illuminating means, preferably in the form of one or more arc lamps or other extended spatially incoherent light source, provides illumination for the sample. The invention generally includes beam splitter and beam combiner means, preferably in the form of a beam splitter/recombiner cube, for splitting the illuminating light into two paths so that it may be directed to the sample through both objective lenses, and for combining observed or emitted light from both objective lenses for recording. A plurality of adjustable mirrors allow the direction of illuminating and/or observed light to and from the objective lenses and image recording means. The image recording means preferably comprises a CCD camera. Means for selectively transmitting and reflecting light of different wavelengths, preferably in the form of one or more dichroic mirrors, are generally included in the invention. Optical path length adjustment means, preferably in the form of a translating stage with one or more suitably positioned mirrors, allows timing  tuning of optical path lengths. Phase compensation means, preferably in the form of chromatic phase compensator plates, may be included for compensation of phase differences between illuminating and observed or emitted light, and/or between different wavelength components within the illumination light and/or within the observed or emitted light. Alignment means for positioning the sample relative to the objective lenses are provided, which preferably include a removable mirror and eyepiece. The invention also may employ vibration isolation supporting means such as a vibration isolated platform or housing.
In a first embodiment of the present invention the two opposing objective lenses are used to a sample simultaneously to obtain two images of the sample, while illuminating light is generally directed to the sample from one objective lens. The two images from the two objective lenses are combined and brought into coincidence on the CCD camera or other imaging means. The optical lengths of the two optical paths from the two objective lenses are adjusted to differ by less than the coherence length of the light emitted from the sample, and preferably by significantly less than a wavelength of the observed or emitted light. The two images will then interfere on the CCD camera to provide sample information. The enhanced depth or Z-resolution information provided by the present invention stems from the interference of these two images when they are combined coherently on the same CCD camera with the length of the two optical paths carefully balanced. While the first embodiment of the present invention is generally described herein in the context of fluorescence microscopy, it will be readily understood by persons skilled in the art that this embodiment is applicable to most other modes of optical microscopy as well, including brightfield, darkfield, and phase contrast microscopies. The first embodiment of the present invention is generally called xe2x80x9cImage Interference Microscopyxe2x80x9d or I2 microscopy, and for convenience and clarity will hereinafter be referred to as the I2M embodiment. The operation of the I2M embodiment of the invention, as well as the other embodiments related below, proceeds in a manner similar to that used in standard optical sectioning microscopy: a series of images of the sample are acquired at different focal planes, with the whole data set being computationally deconvolved to remove the out-of-focus blur by using a previously measured sample of the blur caused by a point source.
In a second embodiment of the invention, which applies primarily to fluorescence or phosphorescence microscopy, illuminating or excitation light from an extended, spatially incoherent source is split by beam splitting means, and used to illuminate the sample from both sides simultaneously through both opposed objective lenses. When the optical path lengths are balanced, the two illumination beams interfere at the focal plane of the two objectives. This narrow interference fringe causes the illumination intensity to vary with depth, Z, in a thin slice or region of the sample surrounding the focal plane. This spatial structure of the illumination light causes a corresponding modulation of the fluorescent emission from the sample, which is the source of the increased Z-direction resolution. In the second embodiment the sample is generally observed through a single objective lens. The second embodiment of the invention is called xe2x80x9cIncoherent Interference Illuminationxe2x80x9d or I3 microscopy, and for convenience and clarity will hereinafter be referred to as the I3M embodiment of the invention.
In a third embodiment of the invention, the I2M embodiment and I3M embodiment are combined and, using essentially the same apparatus, achieve greater Z-direction resolution than is possible with either the I2M or I3M embodiments alone. The third embodiment is hereinafter referred to as the I5 microscopy or I5M embodiment since it involves a combination of the I2M and I3M embodiments. In the I5M embodiment, the sample is observed through both lenses as in the I2M embodiment, while the sample is illuminated from both objective lenses as in the I3M embodiment. The same beam splitter may be used for both the illumination light and the observed light, since the necessary alignment is essentially identical for both.
An object of the invention is to provide a method and apparatus for three-dimensional optical microscopy which provides greatly enhanced depth or Z-direction resolution.
Another object of the invention is to provide a method and apparatus for three-dimensional optical microscopy which has high light throughput.
Another object of the invention is to provide a method and apparatus for three-dimensional optical microscopy which allows high data acquisition speed.
Another object of the invention is to provide a method and apparatus for three-dimensional optical microscopy which does not require use of a coherent light source.
Another object of the invention is to provide a method and apparatus for three-dimensional optical microscopy which does not cause unnecessary bleaching of samples.
Further objects and advantages of the invention will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the invention without placing limitations thereon.